Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal spatial organization. Access to classical and molecular genetics, combined with protein biochemistry, makes this system an ideal one in which to study the molecular basis of development. Structural and functional asymmetry is expressed in each predivisional cell such that upon division, the progeny cells have different developmental programs. An aspect of this asymmetry is the temporally-controlled biogenesis of a flagellum and the chemotaxis machinery at one pole of the predivisional cell. The formation of the flagellum and the chemotaxis apparatus requires the expression of over 40 genes. Our overall goals are to define the molecular mechanisms that regulate when, how much and where to express these genes. We have demonstrated that a large number of these genes are ordered in a trans-acting regulatory hierarchy and that their gene products are localized in the cell. We will attempt to define how this hierarchy functions and how spatial constraints are superimposed on a temporal program of gene expression. To do this we will define individual steps in the hierarchy by using genetics to identify directly interacting pairs of genes, and by using biochemistry to identify and purify specific trans-acting proteins and the cis-acting DNA sequences to which they bind. We will analyze the mechanisms that control temporal transcription by obtaining mutants, both in vivo and in vitro, that alter temporal control and by isolating a possible new RNA polymerase sigma factor for specific fla gene `nif' promoters. We will define the regulatory functions of two sets of overlapping genes in the hierarchy. Finally, we will initiate an investigation of the mechanisms that result in the expression of positional information during the Caulobacter development program by identifying the protein sequences required for cellular localization and by examining the localization of specific mRNAs.